CN108265095B - A kind of15Preparation method of N-stable isotope labeled 5-methyl deoxycytidine - Google Patents

A kind of15Preparation method of N-stable isotope labeled 5-methyl deoxycytidine Download PDF

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CN108265095B
CN108265095B CN201711332504.3A CN201711332504A CN108265095B CN 108265095 B CN108265095 B CN 108265095B CN 201711332504 A CN201711332504 A CN 201711332504A CN 108265095 B CN108265095 B CN 108265095B
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deoxycytidine
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CN108265095A (en
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胡俊杰
范洪波
李衍亮
吴康铭
劳志朗
林勃机
袁美怡
朱丹
梁志辉
曾燕艳
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Dongguan University of Technology
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Abstract

The invention relates to a15The preparation method of the N-stable isotope labeled 5-methyl deoxycytidine comprises the following steps: step 1, selecting a strain; step 2, preparing a catalyst containing15A glucose culture medium and a liquid activation culture medium of ammonium chloride N; step 3, culturing the strains selected in the step 1 in two culture media prepared in the step 2; step 4, extracting and storing bacterial genome DNA and bacterial genome DNA; step 5, CviPI DNA methylase catalytic reaction; step 6, the genome DNA is hydrolyzed,15separating and purifying the N5-methyl deoxycytidine. The invention utilizes microorganisms to synthesize15The N-stable isotope is labeled with 5-methyl deoxycytidine,15the N is easy to mark and has high abundance, the process is simple and the preparation is convenient.

Description

A kind of15Preparation method of N-stable isotope labeled 5-methyl deoxycytidine
Technical Field
The invention relates to the field of microbial fermentation and biological extraction methods, in particular to a method for preparing a stable isotope labeled compound by adopting microbial fermentation.
Background
5-methylcytosine (5-methyl-cytosine) refers to the addition of a methyl group to the carbon atom number 5 of the cytosine base group. 5-methylcytosine has important biological functions, the content of the 5-methylcytosine in genome DNA directly influences the expression and silencing of genes, and the 5-methylcytosine has close connection with reproductive development of organisms, occurrence and deterioration of serious diseases, genetic breeding and the like. Therefore, the content of 5-methylcytosine in genomic DNA of organisms is receiving wide attention from science and technology personnel in environmental toxicology, medicine and pharmacology, genetic breeding and the like. At present, the content of 5-methylcytosine in genome DNA is determined by a plurality of methods, wherein the detection of the cytidine form 5-methyl deoxycytidine of 5-methylcytosine by a high performance liquid chromatography-triple quadrupole mass spectrometer (LC-MS/MS) is proved to be an effective and high-throughput detection method. However, due to the drift of the mass spectrometer signal, the mass spectrometer signal needs to be corrected with stable isotope labeled 5-methyl deoxycytidine.
The production of the 15N stable isotope labeled 5-methyl deoxycytidine can adopt an organic synthesis method, a microbial fermentation preparation method and the like. The synthesis method is simple, but needs a complicated purification process, so that the utilization rate of the 15N raw material is low, and the cost is increased. The direct fermentation method is adopted for production, the target basic groups are enriched in a large amount, and the separation is simple. Chinese patent discloses a method for synthesizing 15N stable isotope labeled arginine (CN 1869244A) by utilizing biological fermentation method,15N-stable isotopically labeled arginine (CN 101235402A),13A method of labeling cellulose (CN 10182445 a) with a C-stable isotope, and the like. However, efficient production by fermentative synthesis using microorganisms has not been found15Patent reports of N-stable isotope labeling of 5-methyl deoxycytidine.
Disclosure of Invention
The invention designs a15A preparation method of N-stable isotope labeled 5-methyl deoxycytidine solves the technical problem of the prior art15The production method of the N-stable isotope labeled 5-methyl deoxycytidine has a fussy purification process, so that15The utilization rate of N raw materials is low, and the cost is increased.
In order to solve the technical problems, the invention adopts the following scheme:
a kind of15The preparation method of the N-stable isotope labeled 5-methyl deoxycytidine comprises the following steps:
step 1, selecting a strain;
step 2, preparing a catalyst containing15A glucose culture medium and a liquid activation culture medium of ammonium chloride N;
step 3, culturing the strains selected in the step 1 in two culture media prepared in the step 2;
step 4, extracting and storing bacterial genome DNA and bacterial genome DNA;
step 5, CviPI DNA methylase catalytic reaction;
step 6, the genome DNA is hydrolyzed,15separating and purifying the N5-methyl deoxycytidine.
Further, Escherichia coli rich in cytosine in the genomic DNA is selected from the strain in the step 1, and is represented by K12.
The used microorganism is escherichia coli and mutant strains thereof, the used strain is conventional strain, and the strain preservation centers are preserved.
Further, the formula of the liquid activation medium in the step 2 is as follows: peptone 0.125% (m/v), yeast extract 0.25% (m/v), disodium hydrogen phosphate 15-35 mM, glucose 3-5 mM in water, and HAC adjusted to pH 6.0.
Further, the step 2 comprises15Glucose medium of ammonium chloride N: m9 minimal medium with glucose as the only carbon source, the formula is: 30-80 mM of sodium chloride, 15-35 mM of potassium dihydrogen sulfate, 50-90 mM of calcium chloride, 1-4 mM of magnesium sulfate, 5-15 mM of glucose and 30-80 mM of 15N ammonium chloride, and the pH value is 6-8.
Further, in the step 3, the 1-ring activated escherichia coli slant seeds in the step 1 are inoculated into the liquid activation culture medium prepared in the step 2, and are subjected to shake culture at 37 ℃ for 12-15 hours, wherein the rotating speed of a shaking table is 160 rpm and the seeds are used as pre-culture seeds; inoculating the pre-cultured seedSeed to step 2 to obtain a seed composition containing15The inoculation amount of the glucose culture medium of the ammonium chloride is 5-8%, the culture is carried out for 48 hours at 37 ℃ by shaking, and the rotating speed of a shaking table is 160 rpm.
Further, in the step 4, the culture obtained in the step 3 is centrifuged for 10 minutes by using a centrifugal force of 300 g, and the cultured bacteria are harvested; extracting genomic DNA by adopting a conventional bacterial genomic DNA extraction kit method, wherein the kit method at least comprises a bacterial lysis step, an RNA degradation step by RNase and a DNA enrichment step by column chromatography;
the genome DNA is stored in a mixed aqueous solution system containing Tris-HCl 10 mM, sodium chloride 50 mM, dithiothreitol 1 mM and magnesium chloride 10 mM and having a pH of 7.9.
Further, in the step 5, 100-200U of CviPI DNA methylase is added into every 100 mg of genomic DNA;
the reaction buffer contained: s-adenosylmethionine 32-64 mM, sodium chloride 50 mM, Tris-HCl 50 mM, dithiothreitol 10 mM, pH 8.5, reacting at 37 ℃ for 4-6 hours; after the reaction, glacial ethanol was added, and 12000 g of the methylated genomic DNA was extracted by centrifugation.
Further, in the step 6, 200-400U nuclease P1, 1-4U phosphodiesterase and 300-700U alkaline sulfatase are added into every 100 mg methylated genomic DNA;
the reaction buffer contained: 0.1M ammonium acetate and 1M ammonium bicarbonate, pH 5.3, reacting at 37 ℃ for 2-6 hours; separating the deoxycytidine mixture formed after hydrolysis by a C18 separation column, collecting eluent, and freeze-drying to obtain the deoxycytidine mixture15N5-methyl deoxycytidine crystal.
A kind of15N5-methyl deoxycytidine crystals, which are produced using the above production method. Obtained by analysis with isotope ratio mass spectrometer15The N stable isotope is used for marking the 5-deoxycytidine crystal,15the abundance of N reaches 99%.
The15The preparation method of the N-stable isotope labeled 5-methyl deoxycytidine has the following beneficial effects:
(1) the invention utilizes microorganisms to synthesize15N-stable isotope labeled 5-methyldiethanol(ii) a salt of an oxycytidine,15the N is easy to mark and has high abundance, the process is simple and the preparation is convenient.
(2) The invention has simple process and low equipment requirement, and is convenient to prepare under laboratory conditions15The N-stable isotope labels 5-methyl deoxycytidine.
Detailed Description
The invention will be further illustrated with reference to the following examples:
example 1:
escherichia coli (Escherichia coli) was used as a starting strain, and the media used included liquid activated medium and glucose medium containing 15N ammonium chloride. The formulation of each medium was as follows:
liquid activation medium: peptone 0.125% (m/v), yeast extract 0.25% (m/v), disodium hydrogen phosphate 15-35 mM, glucose 3-5 mM, and HAC adjusted to pH 6.0.
Comprises15Glucose medium of ammonium chloride N: 30-80 mM of sodium chloride, 15-35 mM of potassium dihydrogen sulfate, 50-90 mM of calcium chloride, 1-4 mM of magnesium sulfate, 5-15 mM of glucose,15ammonium chloride N30-80 mM, pH 6-8.
Inoculating 1 ring of activated slant seeds into a liquid activation culture medium, and performing shake culture at 30 ℃ for 24-36 hours at the oscillation speed of 160 rpm to obtain pre-culture seeds. Inoculating the seeds to a seed container15The inoculum size of the glucose culture medium of the ammonium chloride N is 6 percent, the culture is performed for 24 to 36 hours under the oscillation at the temperature of 30 ℃, the oscillation rotating speed is 160 rpm, and the culture medium is used as the seed for static culture. Will contain15Inoculating a glucose medium-activated seed containing ammonium chloride N15The inoculation amount of the glucose culture medium containing ammonium chloride N is 6 percent, and the culture medium is statically cultured for 48 hours at 30 ℃.
The cultured bacteria were harvested by centrifugation for 10 minutes using a centrifugal force of 300 g. The conventional bacterial genome DNA extraction kit is adopted to extract the genome DNA, and the kit method comprises the steps of bacterial lysis, RNA degradation by RNase, DNA enrichment by column chromatography and the like. The DNA was stored in a Tris-HCl 10 mM, sodium chloride 50 mM, dithiothreitol 1 mM, magnesium chloride 10 mM (pH 7.9) system.
200U of CvipI DNA methylase per 100 mg of genomic DNA was added, the reaction buffer contained: s-adenosylmethionine 64 mM, sodium chloride 50 mM, Tris-HCl 50 mM, dithiothreitol 10 mM, pH 8.5, 37 ℃ for 4 hours. After the reaction, glacial ethanol was added, and 12000 g of the methylated genomic DNA was extracted by centrifugation.
Every 100 mg methylated genome DNA is added with 200U nuclease P1, 1U phosphodiesterase and 300U alkaline sulfatase, and the reaction buffer contains: 0.1M ammonium acetate, 1M ammonium bicarbonate, pH 5.3, at 37 ℃ for 6 hours. Separating the deoxycytidine mixture formed after hydrolysis by a C18 separation column, collecting eluent, and freeze-drying to obtain the deoxycytidine mixture15N5-methyl deoxycytidine crystal. Obtained by analysis with isotope ratio mass spectrometer15The N stable isotope is used for marking the 5-deoxycytidine product,15the abundance of N reaches 99%.
Example 2:
the strain, culture medium and bacterial genomic DNA were extracted in the same manner as in example 1. 100U of CvipI DNA methylase is added to each 100 mg of the obtained Escherichia coli genomic DNA, and the reaction buffer contains: s-adenosylmethionine 32 mM, sodium chloride 50 mM, Tris-HCl 50 mM, dithiothreitol 10 mM, pH 8.5, 37 ℃ for 6 hours. After the reaction, glacial ethanol was added, and 12000 g of the methylated genomic DNA was extracted by centrifugation.
Every 100 mg of methylated genomic DNA is added with 400U nuclease P1, 4U phosphodiesterase and 700U alkaline sulfatase, and the reaction buffer contains: 0.1M ammonium acetate, 1M ammonium bicarbonate, pH 5.3, at 37 ℃ for 2 hours. The deoxycytidine mixture formed after hydrolysis is passed through C18Separating with a separation column, collecting eluate, and freeze drying to obtain the final product15N5-methyl deoxycytidine crystal. Obtained by analysis with isotope ratio mass spectrometer15The N stable isotope labels 5-deoxycytidine products, and the abundance of 15N reaches 99%.
The present invention has been described in connection with the embodiments, and it is obvious that the implementation of the present invention is not limited by the above-mentioned manner, and it is within the protection scope of the present invention as long as various modifications are made by using the method concept and technical scheme of the present invention, or the concept and technical scheme of the present invention is directly applied to other occasions without modification.

Claims (1)

1. A kind of15The preparation method of the N-stable isotope labeled 5-methyl deoxycytidine comprises the following steps:
step 1, selecting a strain; selecting escherichia coli with rich cytosine in genome DNA by the strain in the step 1;
step 2, preparing a catalyst containing15A glucose culture medium and a liquid activation culture medium of ammonium chloride N; the formula of the liquid activation culture medium in the step 2 is as follows: peptone 0.125% (m/v), yeast extract 0.25% (m/v), disodium hydrogen phosphate 15-35 mM, glucose 3-5 mM in water, HAC adjusted to pH 6.0; said step 2 comprises15Glucose medium of ammonium chloride N: m9 minimal medium with glucose as the only carbon source, the formula is: 30-80 mM of sodium chloride, 15-35 mM of potassium dihydrogen sulfate, 50-90 mM of calcium chloride, 1-4 mM of magnesium sulfate, 5-15 mM of glucose,15mixed aqueous solution of 30-80 mM of ammonium chloride N, pH 6-8;
step 3, culturing the strain selected in the step 1 in the two culture media prepared in the step 2; inoculating 1 ring of activated escherichia coli slant seeds obtained in the step 1 into the liquid activation culture medium prepared in the step 2, carrying out shake culture at 37 ℃ for 12-15 hours, wherein the rotating speed of a shaking table is 160 rpm, and taking the seeds as pre-culture seeds; inoculating the pre-cultured seed to the seed containing the seed obtained in step 215The inoculation amount of the glucose culture medium of the ammonium chloride is 5-8%, the culture is carried out for 48 hours at 37 ℃ by oscillation, and the rotating speed of a shaking table is 160 rpm;
step 4, extracting and storing bacterial genome DNA; centrifuging the culture obtained in the step 3 by adopting a centrifugal force of 300 g for 10 minutes, and harvesting the cultured bacteria; extracting genomic DNA by adopting a conventional bacterial genomic DNA extraction kit method, wherein the kit method at least comprises a bacterial lysis step, an RNA degradation step by RNase and a DNA enrichment step by column chromatography; the genome DNA is stored in a mixed aqueous solution system with the pH value of 7.9 and containing 10 mM of Tris-HCl, 50 mM of sodium chloride, 1 mM of dithiothreitol and 10 mM of magnesium chloride;
step 5, CviPI DNA methylase catalytic reaction; adding 100-200U CvipI DNA methylase into every 100 mg of genome DNA; the reaction buffer contained: s-adenosylmethionine 32-64 mM, sodium chloride 50 mM, Tris-HCl 50 mM, dithiothreitol 10 mM, pH 8.5, reacting at 37 ℃ for 4-6 hours; after the reaction is finished, adding glacial ethanol, and centrifuging 12000 g to extract methylated genomic DNA;
step 6, the genome DNA is hydrolyzed,15separating and purifying the N5-methyl deoxycytidine; adding 200-400U nuclease P1, 1-4U phosphodiesterase and 300-700U alkaline sulfatase into every 100 mg of methylated genomic DNA; the reaction buffer contained: 0.1M ammonium acetate and 1M ammonium bicarbonate, pH 5.3, reacting at 37 ℃ for 2-6 hours; separating the deoxycytidine mixture formed after hydrolysis by a C18 separation column, collecting eluent, and freeze-drying to obtain the deoxycytidine mixture15N5-methyl deoxycytidine crystal.
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